JP7360807B2 - Methods to assist in detecting fatigue conditions - Google Patents

Description

The present invention relates to a method for assisting in detecting a state of fatigue in a living body.

You may not feel fatigue even if your body is tired, and conversely, there are diseases such as chronic fatigue syndrome where you feel tired even if your body is not. For this reason, fatigue cannot always be diagnosed accurately only by interviewing the patient. Furthermore, it is obviously not possible to interview animals such as pets or livestock. Therefore, there is a need for a method that can objectively detect fatigue conditions.

A method described in Patent Document 1 is known as a method for detecting fatigue using components in a living body as a biomarker. Patent Document 1 describes various substances as such biomarkers, but also describes the use of alanine, arginine, asparagine, or aspartic acid, which are amino acids that constitute proteins, as biomarkers. However, there is no mention of optical isomers of each amino acid.

WO 2015/030211

An object of the present invention is to provide a novel method that makes it possible to objectively detect a state of fatigue using the concentration of components in a living body as an index.

As a result of intensive research, the inventors of the present application have discovered that a state of fatigue can be detected using the concentration of D-amino acids in vivo or the concentration ratio of D-amino acids and L-amino acids as an indicator, and have completed the present invention.

That is, the present invention provides the following.
(1) A method for assisting in the detection of a fatigued state of a living body, which includes quantifying D-amino acids in a blood sample collected from a living body, and uses the determined concentration of D-amino acids as an index, the method comprising: The D-amino acid is at least one selected from the group consisting of proline, alanine, isoleucine, arginine, valine, threonine, and serine, and at least one selected from the group consisting of proline, alanine, isoleucine, arginine, valine, threonine, and serine. A method in which the concentration of one type of D-amino acid is significantly lower than that of the normal group.
(2) A method for assisting in detecting a fatigue state of a living body, which includes quantifying D-amino acids in a blood sample collected from a living body, and uses the determined concentration of D-amino acids as an index, the method comprising: A method in which the D-form amino acid is methionine, and the D-methionine concentration is significantly higher than that of a normal group.
(3) Including quantifying D-amino acids and L-amino acids in a blood sample collected from a living body, and determining the concentration of each quantified D-amino acid and the concentration of each L-amino acid of the same type as each D-amino acid. A method for assisting in detecting a fatigue state of a living body using the ratio to the concentration as an index, wherein the D-amino acid is at least one selected from the group consisting of proline, isoleucine, alanine, valine, leucine, aspartic acid, and phenylalanine. The concentration of each D-amino acid of at least one type selected from the group consisting of proline, isoleucine, alanine, valine, leucine, aspartic acid, and phenylalanine, and the concentration of each L-amino acid of the same type as each D-amino acid. A method in which the ratio of
(4) Includes quantifying D-amino acids and L-amino acids in a blood sample collected from a living body, and the concentration of each quantified D-amino acid and the concentration of each L-amino acid of the same type as each D-amino acid. A method for assisting in detecting a fatigue state of a living body using the ratio to the concentration as an index, wherein the D-amino acid is methionine, and the ratio to the concentration of L-methionine is significantly higher than that of a normal group. method .

According to the method of the present invention, it is possible to objectively detect a state of fatigue using the concentration or concentration ratio of components actually present in a living body as an index.

In the method of the present invention, the living organisms to be evaluated are preferably mammals and birds; primates such as humans, monkeys and chimpanzees; carnivores such as cats, dogs and hamsters; cows, pigs, horses, sheep, goats, etc. Examples include, but are not limited to, herbivores; rodents such as mice and rats; and birds such as chickens.

In this specification, some of the various amino acids are abbreviated, but their official names are as follows.
(abbreviation) (official name)
Ala Alanine Arg Arginine Asn Asparagine Gln Glutamine Gly Glycine His Histidine Ile Isoleucine Leu Leucine Lys Lysine Met Methionine Phe Phenylalanine Pro Proline Ser Serine Thr Threonine Trp Tryptophan Tyr Tyrosine Val Valine

The biological sample is not particularly limited as long as it is a sample collected from a living body, but body fluids such as blood, urine, and saliva are preferred, and blood is particularly preferred. The blood sample may be whole blood, serum, or plasma.

The "fatigue state" detected by the method of the present invention includes both a state of mental fatigue and a state of physical fatigue. In the following examples, it is considered that a state of mental fatigue is mainly detected.

In the method of the present invention, at least D-amino acids in a biological sample are quantified. In one embodiment, L-amino acids are also quantified. Previously, it was thought that only L-amino acids made up living proteins, but in recent years, analytical technology has improved and it is now possible to distinguish between D-amino acids and L-amino acids and quantify them. It has become clear that D-amino acids are also present in living organisms. A method for distinguishing and quantifying amino acids into D-form and L-form is known (WO 2017/057433), and is specifically described in the Examples below.

In the first embodiment of the present invention, D-amino acids in a biological sample are quantified, and a state of fatigue is detected using the determined concentration of D-amino acids as an index. The D-amino acid to be quantified is preferably at least one selected from the group consisting of proline, alanine, isoleucine, arginine, valine, methionine, threonine, and serine. In the Examples below, these D-amino acids were those for which a statistically significant difference was observed between the fatigue group and the normal group. Among these, the concentration of proline, alanine, isoleucine, arginine, valine, threonine, and serine is significantly lower in the fatigue group than in the normal group, and the concentration of methionine is significantly higher in the fatigue group than in the normal group. Table 1 below shows the ratio of the average value of the concentration of D-amino acid in the fatigue group to the average value in the normal group (fatigue group/normal group), which was measured in the following example.

The fatigue group/normal group ratio value shown in Table 1 or a value near it is set as the cutoff value, and if the concentration of the D-amino acid is lower than the cutoff value (or higher in the case of methionine), It can be determined that the patient is in a "fatigued state". In this case, the cutoff value varies to some extent depending on the group to be tested, so it can be set appropriately, for example, about ±30% of the value shown in Table 1 above, preferably about ±20%, and Preferably, it can be set within a range of about ±10%.

In the second embodiment of the present invention, D-amino acids and L-amino acids in a biological sample are quantified, and the concentration of each quantified D-amino acid and the concentration of each L-amino acid of the same type as each D-amino acid are determined. (hereinafter sometimes referred to as the "D/L ratio" for convenience) is used as an index. The amino acid to be quantified is preferably at least one selected from the group consisting of proline, isoleucine, alanine, valine, methionine, leucine, asparagine, and phenylalanine. In the following examples, statistically significant differences were observed in the D/L ratios of these amino acids between the fatigue group and the normal group. Among these, the D/L ratio of proline, isoleucine, alanine, valine, leucine, asparagine, and phenylalanine was significantly lower in the fatigue group than in the normal group, and the D/L ratio of methionine was significantly lower in the fatigue group than in the normal group. is also significantly higher. Table 2 below shows the ratio between the average value of the D/L ratio of each amino acid in the fatigue group and the average value of the normal group (fatigue group/normal group), which were measured in the following examples.

The value of the fatigue group/normal group ratio shown in Table 2 or a value near it is set as the cutoff value, and if the D/L ratio is lower than the cutoff value (or higher in the case of methionine), It can be determined that the patient is in a state of fatigue. In this case, the cutoff value varies to some extent depending on the group to be tested, so it can be set appropriately, for example, about ±30% of the value shown in Table 2 above, preferably about ±20%, and Preferably, it can be set within a range of about ±10%.

Hereinafter, the present invention will be specifically explained based on Examples. However, the present invention is not limited to the following examples.

Example 1
Six-week-old Wistar male rats (Japan SLC Co., Ltd.) were purchased and used at seven weeks of age after preliminary breeding. Six rats in the fatigue group were kept for 5 days from Day 1 to Day 6 under free feeding and drinking conditions in a cage filled with water adjusted to 23±1° C. at a height of 1.5 cm from the bottom. The water in the cage was changed daily. On the other hand, six rats in the normal group were kept in cages with bedding (Pepper Clean, Pepperette Co., Ltd.) under free feeding and drinking water conditions for 5 days. Whole blood was collected from the abdominal aorta under isoflurane anesthesia using a needle and a syringe containing an appropriate amount of heparin sodium in the barrel. The obtained blood was centrifuged at 1870×g for 10 minutes at 4° C. to separate plasma. The obtained plasma was stored at -20°C or below until analysis.

- Preparation of sample To 20 μL of rat plasma, 20 μL of an internal standard solution labeled with a stable isotope of amino acids was added, and then 40 μL of acetonitrile was added, followed by centrifugation at 20° C. and 20,000×g for 10 minutes, and the supernatant was collected. Add 2.5 mg/mL (R)-4-nitrophenyl-N-[2'-(diethylamino)-6,6'-dimethyl-[1,1'-biphenyl]-2 of derivatization reagent solution to 10 μL of supernatant. -ylcarbamate hydrochloride [(R)-BiAc]] was added and reacted at 55°C for 10 minutes, then 100 μL of 0.1% formic acid aqueous solution was added, stirred, and subjected to liquid chromatography/triple quadruplex. 5 μL was used for polar mass spectrometry.

-Separation by liquid chromatography Nexera (registered trademark) manufactured by Shimadzu Corporation was used as a liquid chromatography system (LC). The temperature of the analytical column was set at 40°C. The analytical columns, eluents, and flow rates used are as shown below.

(analytical column)
Triart Phenyl manufactured by YMC (2.1mm x 75mm 1.9μm)
(eluent)
A eluent: 10 mmol/L ammonium formate/0.1% formic acid/water B eluent: 95% acetonitrile/water (flow rate)
0.4mL/min

For separation by LC, an analytical sample was introduced into an analytical column equilibrated with 86% A eluent and 14% B eluent. The B eluent was ramped linearly to 16% at 3 minutes, then 33% at 14.3 minutes, and 45% at 17 minutes to elute the amino acids. Then, from 17.1 minutes, the B eluent was increased to 90%, the analytical column was washed for about 1 minute until 18 minutes, and then equilibrated at the initial state (A eluent 86%, B eluent 14%) until 20 minutes. It became.

- Detection using a mass spectrometer The mass spectrometer used was Triple Quad 6500 (registered trademark) manufactured by Sciex, and electrospray ionization (positive ions) and SRM mode were used. Mass spectrometry was performed under the following conditions.

(Ion source parameters)
Curtain:40
Collision Gas Setting:8
Ion Spray Voltage: 4500
Temperature:600
Ion Source Gas 1:70
Ion Source Gas 2:70
Interface Heater:ON

The L-amino acid was set at m+2 of the protonated molecule derived from the isotope. Furthermore, L-His, L-Arg, and L-Lys were detected as divalent ions. The IS of each amino acid indicates the internal standard substance used in quantifying each D-form and L-form amino acid.

- Quantification method Blood concentrations of various amino acids were determined using the peak area of the chromatogram of each amino acid separated from LC and by selecting an internal standard method. That is, the concentrations of amino acids with known concentrations were plotted on the x-axis and the peak area ratios (peak area values of various amino acids/peak area values of internal standard substances) were plotted on the y-axis, and a calibration curve was created by the least squares method. Next, the peak area ratio (peak area value of various amino acids/peak area value of internal standard substance) was calculated in the same way from the chromatograms of each amino acid in the rat samples of the normal group and the fatigue group, and the peak area ratio of each amino acid was calculated from the calibration curve. The concentration was calculated.

Analyst (registered trademark) manufactured by Sciex was used as data acquisition and analysis software for LC-MS/MS, and quantitative values were calculated from the chromatogram of each amino acid in the sample.

Statistical analysis was performed using SAS9.4 (SAS Institute Inc.) and its linked system EXSUS ver. A t-test was performed using 8.1 (CAC Croix Co., Ltd.).

Table 4 shows the results of measuring the blood D-amino acid concentration in Example 1. The numerical values in the table represent the average value and standard error (μM) of 6 cases. "*" in the table indicates a statistically significant difference (P<0.05) compared to the normal group.

The concentrations of arginine, serine, threonine, alanine, proline, valine, and isoleucine in the normal group were 1.92, 1.48, 0.10, 7.21, 0.40, 0.14, and 0.21 μM, respectively. . In contrast, the concentrations of arginine, serine, threonine, alanine, proline, valine, and isoleucine in the fatigue group were 1.14, 1.13, 0.07, 2.49, 0.10, 0.09, and 0.09, respectively. It was 12 μM, which was significantly decreased compared to the normal group. On the other hand, the methionine concentration in the normal group was 3.02 μM. On the other hand, in the fatigue group, it was 4.64 μM, which was significantly increased compared to the normal group.

Table 5 shows the ratio between the blood D-amino acid concentration and the corresponding L-amino acid concentration in Example 1. The numerical values in the table indicate the average value and standard error (%) of 6 cases. "*" in the table indicates a statistically significant difference (P<0.05) compared to the normal group. The ratios of the D-amino acid concentrations and the corresponding L-amino acid concentrations of asparagine, alanine, proline, valine, isoleucine, leucine, and phenylalanine in the normal group were 2.90, 2.31, 0.26, and 0.06, respectively. They were 0.19, 0.29 and 0.08%. In contrast, the ratios of the D-amino acid concentrations of asparagine, alanine, proline, valine, isoleucine, leucine, and phenylalanine to the corresponding L-amino acid concentrations in the fatigue group were 2.26, 1.03, and 0.08, respectively. They were 0.03, 0.08, 0.16 and 0.06%, which were significantly decreased compared to the normal group. On the other hand, the ratio of D-methionine concentration to L-methionine concentration in the normal group was 5.16%. On the other hand, in the fatigue group, it was 9.35%, which was significantly increased compared to the normal group.

This result showed that the D-amino acid of the present invention or the ratio of the D-amino acid concentration to the corresponding L-amino acid concentration changes in a fatigued state.

Claims (4)

A method for assisting in detecting a fatigue state of a living body, the method comprising quantifying D-amino acids in a blood sample collected from a living body, and using the determined concentration of D-amino acids as an index. is at least one selected from the group consisting of proline, alanine, isoleucine, arginine, valine, threonine and serine, and at least one selected from the group consisting of proline, alanine, isoleucine, arginine, valine, threonine and serine. A method in which the D-amino acid concentration is significantly lower than that of the normal group. A method for assisting in detecting a fatigue state of a living body, the method comprising quantifying D-amino acids in a blood sample collected from a living body, and using the determined concentration of D-amino acids as an index. is methionine, and the D-methionine concentration is significantly higher than that of the normal group. The method includes quantifying D-amino acids and L-amino acids in a blood sample collected from a living body, and the concentration of each quantified D-amino acid and the concentration of each L-amino acid of the same type as each D-amino acid. A method for assisting in detecting a fatigue state of a living body using ratio as an index, wherein the D-amino acid is at least one selected from the group consisting of proline, isoleucine, alanine, valine, leucine, aspartic acid, and phenylalanine. The ratio of the concentration of each D-amino acid of at least one selected from the group consisting of proline, isoleucine, alanine, valine, leucine, aspartic acid, and phenylalanine to the concentration of each L-amino acid of the same type as each D-amino acid. but significantly lower than the normal group. The method includes quantifying D-amino acids and L-amino acids in a blood sample collected from a living body, and the concentration of each quantified D-amino acid and the concentration of each L-amino acid of the same type as each D-amino acid. A method for assisting in detecting a state of fatigue in a living body using the ratio as an index, wherein the D-amino acid is methionine, and the ratio to the concentration of L-methionine is significantly higher than in a normal group.